Computational protocol: Real-Time Imaging of Resident T Cells in Human Lung and Ovarian Carcinomas Reveals How Different Tumor Microenvironments Control T Lymphocyte Migration

Similar protocols

Protocol publication

[…] Image analysis was performed at the Cochin Imaging Facility (Institut Cochin, Paris). A 3D image analysis was performed on x, y, and z planes using Imaris 7.4 (Bitplane AG). First, superficial planes from top of the slice to 15 μm in depth were removed to exclude T cells located near the cut surface. Cellular motility parameters were then calculated using Imaris. Tracks >10% of the total recording time were included in the analysis. The straightness value was calculated as the ratio of the distance from origin to the total distance traveled. To reveal the relationship between CD8 T cell motility and the tumor structure (tumor islets and collagen network), confocal time-lapse images of T cells were superimposed onto the corresponding SHG and EpCAM images. CD8 T cells localized in the stroma were distinguished from those infiltrated in tumor cell nests by looking at individual planes along the z axis. Videos and images were made by compressing the z information into a single image using Imaris. When a drift in the x, y dimension was noticed, it was corrected using the “Correct 3D Drift” plug-in in ImageJ. For the automated detection of resident CD8 T cells in different tumor areas (stroma, tumor islets, loose, and dense collagen regions identified by visual inspection of SHG images), we used the ImageJ software. First, fluorescent images were thresholded and converted to binary images. Angles between the cell trajectory vectors, which are the connecting lines between starting points and end points of each track, and tumor-stroma boundaries were calculated using Image J software. Only the cells positioned within a maximum distance of 100 μm from the tumor-stroma interfaces were included in further analysis. Distances between collagen fibers were determined by using the “point to point distance measurement” function of Imaris. […]

Pipeline specifications

Software tools Imaris, Correct 3D Drift, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Diseases Lung Neoplasms, Neoplasms, Ovarian Neoplasms